Chronic hepatitis B virus (HBV) infection affects approximately 350 million people worldwide and is a significant risk factor for severe liver disease, including hepatocellular carcinoma (HCC). The role of HBV in mediating carcinogenesis is complex and includes integration of viral DNA into host chromosomes, the host immune system, and expression of the viral regulatory HBx protein. Our laboratory studies HBx, a small protein required for virus replication in vivo that is also involved in carcinogenesis We and others have shown that the interaction of HBx with cellular damaged DNA binding protein (DDB1) is critical for HBV replication. However, the functional implications of this interaction to viral replication and pathogenesis remain elusive. DDB1 is an adaptor protein for the Cul4-DDB1 E3 ligase complex, and acts in part by recruiting cellular DDB1- Cullin Accessory Factors (DCAFs) that act as substrate receptors to recruit additional cellular targets for ubiquitination and proteasome degradation. In this manner, DDB1 regulates diverse cellular functions such as DNA replication, cell cycle, innate immunity, and damaged DNA repair. We hypothesize that HBx acts as a viral DCAF, and displaces one or more cellular DCAFs from the Cul4-DDB1 complex, thereby modifying the cellular environment to favor virus replication. Human liver chimeric mice, in which human hepatocytes are engrafted into immunodeficient mice, provide the best, most biologically relevant model system to investigate the impact of HBV replication on Cul4-DDB1-DCAF complexes. In Specific Aim 1, an immunoprecipitation/mass spectrometry (MS) approach will be used to identify the Cul4-DDB1-DCAF profile of uninfected human liver, providing new insights into DDB1 pathways important to normal liver physiology. This will be compared to the Cul4-DDB1-DCAF profile from HBV-infected hepatocytes originating from the same donor liver, using quantitative MS analyses to compare relative binding levels of DCAFs to DDB1. In Specific Aim 2, the importance of HBx-DDB1 binding to DDB1 pathways will be investigated. To determine whether HBV activates the DNA damage response (DDR) as part of its acute replication strategy, uninfected and HBV- infected human liver tissue from chimeric mice will be examined by western blot for evidence of the DDR marker, phosphorylated histone H2AX. To determine whether HBx-inactivation of interferon signaling occurs through HBx-DDB1 binding, the ability of HBx mutants that cannot bind DDB1 to inhibit interferon signaling will be evaluated in vitro. Additionally, MS data will be mined for evidence that HBV replication induces recruitment of immune regulatory proteins to the Cul4-DDB1 complex. In summary, viruses are adept at targeting cellular pathways that benefit viral replication. The proposed studies will elucidate the impact of HBV replication on the cellular ubiquitination and degradation machinery using the innovative human liver chimeric animal model. This important new knowledge will reveal virus-induced changes at the post-translational level, and is predicted to provide mechanistic insight into new strategies through which to treat chronic HBV infection and prevent liver disease.